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Historically students of craniology believed, erroneously, that only the active processes of surface deposition and resorption and of interstitial expansion were involved in skull growth. The introduction of the method of functional cranial analysis placed primary emphasis on the morphogenetic role of the functional matrix. The two principal types of functional matrices, the periosteal and the capsular, are defined. The former alter the size and shape of the skeletal tissues while the latter alter spatial position. The majority of facial skeletal growth is shown to be due to the passive translation of the skeletal tissues within the orofacial capsule, responding to the prior and primary volumetric expansion of the oro-nasopharyngeal functioning spaces, acting as capsular matrices.

The purpose of this study was to clarify the differences in the nature of craniofacial growth between subjects with normal occlusion and patients with unilateral cleft lip and palate (CLP) in terms of the size, shape and principal growth direction of craniofacial skeleton using finite element method (FEM). Lateral cephalograms were taken of 40 subjects as normal group and 178 patients as CLP group. These subjects were divided into seven developmental ages of 4-, 6-, 8-, 10-, 12-, 14- and 18-year-old. For the finite element analysis, the craniofacial complex was discretized into seven structures or elements with three nodal points in each after tracing each lateral cephalogram on acetate paper. For each stage, the growth parameters in CLP group were compared to those in normal group. The growth of upper facial skeleton and maxillary complex was more remarkably inhibited in CLP than in normal group. Especially, the growth inhibition of posterior maxillary complex in a vertical direction was remarkable in CLP group at any ages. Difference in the size and shape of entire mandibular skeleton between CLP and normal groups was not apparent. It is suggested that grow timing and peak velocity, an essential and key determinant to the success in orthodontic treatment, have been clarified in this study more clearly than in previous studies. It is hopefully anticipated to explore some key determinants to predict individual growth of the craniofacial skeleton near future.

The purpose of this study was to elucidate the nature of human craniofacial growth by means of finite element method (FEM), and to compare the results with the current concept derived from morphometric studies with roentgenographic cephalometry (RCM). Lateral cephalograms were taken of 20 males and 20 females, and traced on acetate paper. On the tracing, the craniofacial complex was divided into seven areas or elements. Growth analysis was executed with FEM for changes in the size and shape of each area during six developmental periods from 4 to 18 years old. The size change in the male group exhibited a peak from 10 to 12 years old for the cranial base and upper facial skeleton, from 8 to 10 years old for the maxillary complex, and from 12 to 14 years old for the mandible. On the other hand, in the females, the size change was almost invariable from 4 to 12 years old and completed suddenly at 12 years old, which was earlier by a few years than in the males. An interesting finding is that changes in the size and shape were the most substantial in the maxillary complex, followed by those in the mandible, and the smallest in the cranial base and upper facial skeleton. It is shown that growth of the cranial base lasted up to 14 years old and the maxillary complex exhibited the greatest growth changes in the size among various anatomic structures. These findings may provide a new concept of human craniofacial growth, somewhat different from the previously established principles by RCM.

In two interrelated articles, the current revision of the functional matrix hypothesis extends to a reconsideration of the relative roles of genomic and of epigenetic processes and mechanisms in the regulation (control, causation) of craniofacial growth and development. The dialectical method was chosen to analyze this matter, because it explicitly provides for the fuller presentation of a genomic thesis, an epigenetic antithesis, and a resolving synthesis. The later two are presented here, where the synthesis suggests that both genomic and epigenetic factors are necessary causes, that neither alone is also a sufficient cause, and that only the two, interacting together, furnish both the necessary and sufficient cause(s) of ontogenesis. This article also provides a comprehensive bibliography that introduces the several new, and still evolving, disciplines that may provide alternative viewpoints capable of resolving this continuing controversy; repetition of the present theoretical bases for the arguments on both sides of these questions seems nonproductive. In their place, it is suggested that the group of disciplines, broadly termed Complexity, would most likely amply repay deeper consideration and application in the study of ontogenesis.

Although the initial versions of the functional matrix hypothesis (FMH) theoretically posited the ontogenetic primacy of "function," it is only in recent years that advances in the morphogenetic, engineering, and computer sciences provided an integrated experimental and numerical data base that permitted recent significant revisions of the FMH--revisions that strongly support the primary role of function in craniofacial growth and development. Acknowledging that the currently dominant scientific paradigm suggests that genomic, instead of epigenetic (functional) factors, regulate (cause, control) such growth, an analysis of this continuing controversy was deemed useful. Accordingly the method of dialectical analysis, is employed, stating a thesis, an antithesis, and a resolving synthesis based primarily on an extensive review of the pertinent current literature. This article extensively reviews the genomic hypothesis and offers a critique intended to remove some of the unintentional conceptual obscurantism that has recently come to surround it.

The following case report describes the expanding role of pediatric dentists in treating children with craniofacial pathology. Retinoblastoma is the most common intraocular malignancy in childhood and is approximately the tenth most common pediatric cancer in the United States. Treatment consists of enucleation, or removal of the entire globe followed by placement of orbital implants. Un-restored anopthalmic sockets exhibit growth retardation and can lead to facial disfigurement. Maxillofacial prosthetic (MFP) rehabilitation can be especially challenging in younger, pre-cooperative or behaviorally compromised children and requires the skills and participation of a pediatric dental specialist as part of the MFP team. The following case report involving a 3 yr-old girl with retinoblastoma describes such challenges. The objective of the maxillofacial prosthetic team was to provide custom-built, acrylic, bilateral ocular prostheses in as comfortable and atraumatic manner as possible. The case was a success and underscores the value of a multidisciplinary dental approach for the treatment of children with very special needs.

Intercellular gap junctions permit bone cells to intercellularly transmit, and subsequently process, periosteal functional matrix information, after its initial intracellular mechanotransduction. In addition, gap junctions, as electrical synapses, underlie the organization of bone tissue as a connected cellular network, and the fact that all bone adaptation processes are multicellular. The structural and operational characteristics of such biologic networks are outlined and their specific bone cell attributes described. Specifically, bone is "tuned" to the precise frequencies of skeletal muscle activity. The inclusion of the concepts and databases that are related to the intracellular and intercellular bone cell mechanisms and processes of mechanotransduction and the organization of bone as a biologic connected cellular network permit revision of the functional matrix hypothesis, which offers an explanatory chain, extending from the epigenetic event of muscle contraction hierarchically downward to the regulation of the bone cell genome.

The periodic incorporation of advances in the biomedical, bioengineering, and computer sciences allow the creation of increasingly more comprehensive revisions of the functional matrix hypothesis. Inclusion of two topics, (1) the mechanisms of cellular mechanotransduction, and (2) biologic network theory, permit this latest revision; presented here in two interrelated articles. In this first article, the several possible types of intracellular processes of mechanotransduction are described. These translate the informational content of a periosteal functional matrix stimulus into a skeletal unit (bone) cell signal. The correlation between the strengths of the endogenous electrical fields produced by muscle skeletal muscle activity, and those to which bone cells maximally respond are stressed. Further, a physical chain of macromolecular levers, connecting the extracellular matrix to the bone cell genome is described, suggesting another means of epigenetic regulation of the bone cell genome, including its phenotypic expression.

Conventional methods of roentgenographic cephalometry will yield differences in interpretation of growth or treatment changes depending on which methods of superimposition are used. The finite element method of analysis has been reported to have significant advantages since it does not require a reference frame to describe the changes that have taken place. This article describes the growth of a patient with a Class II, Division 1 malocclusion during approximately 2 years of orthodontic treatment with the use of conventional cephalometric growth and static analyses, as well as the finite element method. Thirty cephalometric points were digitized on the pretreatment and the posttreatment lateral cephalograms of a patient undergoing orthodontic treatment between the ages of 10 and 12 years. The finite element method provided a reference frame invariant description of the size, shape, change, and rotation of each of the 12 finite elements representing different anatomic structures.

A random flap can be constructed, its circulation determined, and the ischemic portion identified. Left untreated for a period, the critical ischemia time, the ischemic portion will die and is clinically recognized several days later. What is not known is when this tissue, destined to die, actually dies. To ascertain this time, we compared the percent necrosis of a distal 3 x 3 cm segment of a 10 x 3 cm reverse McFarlane random flap with a known distribution of necrosis to the percent necrosis of the distal 3 x 3 cm of full-thickness skin grafts taken from a similar reverse McFarlane flap at 0, 4, 8, 12, and 16 hours after pedicle construction. Implicit in this experiment is the assumption that necrosis of the full-thickness skin grafts in excess of that of control animals represented skin no longer viable. Sometime between 8 and 12 hours, the percent necrosis of the full-thickness skin grafts surpassed that of the control, and it was concluded that this graft was dead prior to grafting. Thus it is suggested that critical ischemia time and death of the flap tissue are nearly identical, and the latter occurs at between 8 and 12 hours.

The application of the concepts of continuum mechanics and of the numerical techniques of the finite element method permits the development of a new and potentially clinically useful method of describing craniofacial skeletal growth. This new method differs from those associated with customary roentgenographic cephalometry in that its descriptions and analyses are invariant; that is, they are independent of any method of registration and superimposition. Such invariance avoids the principal geometric constraint explicit in all analytical methods associated with conventional roentgenographic cephalometry. The conceptual and mathematical bases of the finite element method (FEM) are presented and illustrated by the numerical and graphic descriptions of the two-dimensional growth of the rat skull, for which two sets of longitudinal growth data are used. In practice, the FEM permits analysis of the skull at a scale significantly finer than previously possible, by considering cranial structure as consisting of a relatively large number of contiguous finite elements. For each such element, independently, it is then possible to describe and depict both the magnitude and the direction of temporal size and shape changes occurring in that element relative to itself at some initial time. It is emphasized that such descriptions are completely independent of any local reference frame.

Methods for the mathematical description of growth are reviewed and extended. The main mode of description is that of identifying the material point paths (in space-time) of the mass points which make up the animal or plant. The spatial location and mass of the elementary particles of the animal are considered to be continuous in space-time. Discontinuities are allowed in displacement functions to allow for slip, as in a knee joint; discontinuities are also allowed in material properties in the sense of sudden adhesion or non-adhesion of surfaces and in the topology, such as the completion or opening of a ring. When cells proliferate or atrophy throughout the tissue growth is considered as distributed sources or sinks in space-time. Deposition or resorption on the surface of a bone is considered to be a surface distribution of mass sources or sinks in space-time. The theoretical framework is illustrated by idealized examples of mathematical descriptions involved.

Recent data on shark skeletal tissues have been reviewed. It is now reasonably certain that shark teeth and denticles are covered with a true ectodermal enamel, although the outer layer of these structures is structurally complex, consisting of both calcified ectodermal enamel and uncalcified areas of ectomesenchymal origin. The intradermal base of these structures most probably consists of acellular bone. The structural array of apatite crystallites in the teeth seems strongly correlated with the specific shape and function of individual teeth. The calcified cartilage of sharks differs significantly from that of other vertebrates, not only in its composition, but in the fact that the areas of calcification are composed of many vital and non-hypertrophic cells. Recent studies of the mineralization processes of other vertebrate tissues suggest a possible explanation for the classically described differential patterns of calcification of shark cartilages, but the specific details in elasmobranchs are as yet unknown, as indeed are many other aspects of their skeletal tissues whose future investigation would surely be useful in the elucidation of the general processes of vertebrate skeletal tissue mineralization.

Summary The factors relating to the development of fenestration of the vertical-lateral plate of the maximillary bone in the rabbit was studied by ontogenetic, comparative, phylogenetic and experimental materials. Normal developmental sequences showed that the trabecular bone pattern of the vertical plate is produced by the processes of periosteal slippage related to the upward growth of the nasal roof and to the structure and disposition of the snout periosteum. Experimental surgical data and the data in the literature suggest that fenestration of the vertical plate reflects the normal lack of transmission of masticatory forces through the vertical plate. Such fenestrations tend to be replaced by compact bone, and compact bone tends to be replaced by fenestrated bone when mastication force transmission is altered. The evolution of leporid fenestration and its differential expression in recent genera demonstrate the same principle. An analysis of snout growth and of cranial structure is given that support the hypothesis presented. Zusammenfassung Eine funktionelle Analyse der fenestrierten Maxilla des Kaninchens (Oryctolagus cuniculus) Einflusse auf die Entwicklung der Fenestration der Tabula externa maxillae des Kaninchens wurden ontogenetisch, vergleichend, phylogenetisch und experimentell untersucht. Die Ontogenese zeigt, das das trabekulare Muster durch Gleitbewegungen des Periosts zustandekommt, die mit dem Dorsal-wachstum des Nasendaches und der Struktur und Anlage der rostralen Knochenhaut zusammenhangen. Chirurgisch-experimentelle Daten und Literaturangaben legen nahe, das diese Fenestration das Fehlen von Kaudrucken widerspiegelt. Bei Anderungen des Kaudrucks sind Wechsel von Fenestrationen in kompakte Knochensubstanz und umgekehrt bekannt. Die Entwicklung der Fenestration bei Leporiden und ihre Auspragung bei rezenten Formen demonstrieren dasselbe Prinzip. Eine Analyse des Wachstums des Splanchnocranium und anderer Schadelstrukturen wird durchgefuhrt; sie untermauert diese Hypothese. Resume Analyse fonctionnelle de l' maxillaire fenětre du Lapin (Oryctolagus cuniculus) Les facteurs qui regissent le developpement de la feněration de la lame verticale laterale du maxillaire chez le Lapin ont eteetudies sur les plans onto-genique, comparatif, phylogenique et experimental. Les sequences du developpement normal ont montre que ľorganisation trabeculaire de ľos de cette lame est produite par les processus de ľexpansion du perioste lies aľaccroissement du plafond du nez en direction dorsale ainsi qua la structure et a la disposition du perioste de la region. Les resultats de la chirurgie experimentale et les donnees de la litterature suggerent que la feneration de la lame verticale traduit ľabsence normale de transmission des forces de la mastication a travers cette lame. De telles fenerations tendent aetre remplacees par de ľos compact et ľos compact tend au contraire a ceder la place a de ľos fenetre quand la transmission des forces de mastication est alteree. Ľevolution de la fenetration chez les Leporides et son expression differente dans les genres regents met en evidence le meme principe. Une analyse de la croissance du museau et de la structure crâniennes confirme ľhypothese exposee. Resumen Un analisis funcional del hueso maxilar fenestrado del conejo (Oryctolagus cuniculus) En material ontogenyetico, comparado, filogenetico y experimental, se estudiaron los factores que conducen al desarrollo de la fenestracion de la lamina vertico-lateral del hueso maxilar en el conejo. Secuencias normales del desarrollo demostraron que la disposicion de las trabeculas oseas en la lamina vertical se produce debido al proceso de deslizamiento del periostio relacionado con el crecimiento hacia arriba del techo nasal y con la estructura y disposicion del periostico del hocico. Los resultados de experimentos quirurgicos y los datos en la bibliografia sugieren que la fenestracion de la placa vertical refleja simplemente la falta normal de la trasmision de fuerzas masticatorias a travel de esta placa. Tal fenestracion tiende a reemplazarse por hueso solido, y hueso solido tiende a reemplazarse por hueso fenestrado, de acuerdo con alteraciones en la trasmision de las fuerzas de la masticacion. La evolucion de la fenestracion en el conejo y su grado diferente en generaciones recientes obedecen al mismo principio. Se presenta un analisis del crecimiento del hocico y de las estructuras de la cabeza osea, que concuerdan con esta hipotesis.

An allometric centered model of craniofacial growth was tested by several computer-assisted statistical methods on the pure longitudinal growth data of twenty-four close-bred female rats and on cross-sectional human cranial growth data. The study demonstrated that such a model was heuristic and, being incapable of exact definition, was deemed inappropriate for further use in modeling of craniofacial skeletal growth. The necessity for vigorous testing of any hypothesis concerning the modeling of craniofacial growth is stressed.

Much of the continuing controversy concerning the roles of genomic and nongenomic (epigenetic) processes in the regulation (causation) of growth is resolved by an analysis of the several types of causation. It can be shown that the combination of genomic and epigenetic factors is a necessary cause of craniofacial growth. A review of some recent literature serves to clarify this conclusion which is of potential clinical use since therapeutic intervention is always an epigenetic event.

Recurrent reports by others of posteruptive dimensional increase of the crowns of rat molar teeth were analyzed in the context of our present study of occlusal attrition, continuous eruption and alteration of the occlusal planes of rat maxillary molar teeth with age. Marked attrition of the anatomical crowns occurs, together with a considerable continuous eruption that increasingly brings the markedly convex mesial root of the maxillary first molar into the clinical crown. Further, a slight change in occlusal plane occurs. Previous workers used standardized planes of orientation and of registration prior to measurement, which masked the phenomena mentioned above. They thus mistakenly reported increased coronal dimensions.

The positional stability of the zygomatic arch of the rat skull in relation to the parietal bone, the basisphenoid bone and the length axis of the skull, as previously demonstrated in rats older than 14 days, does not exist to the same degree in younger rats. These conclusions have been drawn from a study of a sample of rats, aged 0, 4, 7 and 14 days. These 4 groups of rats have been x-rayed after sacrifice, and from the x-ray photographs, quantitative expressions of angular values between the mentioned structures have been obtained. The most important result is, however, considered to be, that a full description of the behavior of the external cranial frame of the rat skull is now available for further studies on the process of orthocephalization. res during orthocephalization of the rat skull.

This study suggests that: 1. The porous plastic material tested is compatible with and showed no undesirable reactions in subcutaneous, intracerebral, intramuscular, and especially, intraosseous sites. 2. By standardizing porosity (e.g., a desired hole, any dimension, uniformly distributed) with the implant material (P.P.M.M.), one can predict with a degree of certainty that connective-tissue (collagen fibers) or hard-tissue (osteogenesis and reparative bone) ingrowth into these micropores will definitely occur. 3. The various shapes employed for implantation of the plastic material were not carcinogenic and did not cause a malignant reaction in any implanted region. 4. A close "amalgamation" with adjacent tissues was a result regardless of the implant site (e.g., intramuscular, intracerebral, or subcutaneous). Intraosseously, there was a variation. Depending upon the pore size, one observes either connective tissue or osseous ingrowth into the micropores. At a 100 micron pore size (B material), connective tissue ingrowth into the P.P.M.M. was seen. At the 450 micron size (E material), osseous tissue ingrowth into the P.P.M.M. was clearly observed. 5. The P.P.M.M. implant material can be used as a tooth replacement (e.g., for a root) at a small pore size (100 micron) and as a bone replacement (e.g., for bony defects) at a larger pore size (450 micron). 6. To secure vascularization of the implant material, porosity throughout is indicated at a large pore size. Surface porosity regardless of size will encourage only soft-tissue ingrowth into the implant material. 7. Any hard-tissue size or shape can be duplicated with the material, at any pore size, with compatibility to the adjacent tissue, regardless of the region of placement in the body. 8. There is a possibility of bone "induction" or stimulation of bone growth in predetermined directions with all its implications (e.g., periodontal disease). The replacement and repair of any hard tissue in the body (e.g., bone and teeth) are possibilities.

Summary The growth of the human mandible in a series of orthodontically normal children was examined by methods designed to demonstrate both the vectors of active and passive growth and the logarithmic attributes of such growth. Both methods are described and illustrated. The individual differences in the vectorial properties of active and passive growth during relatively short periods of time suggest that is is hazardous to predict such growth increments within clinically useful tolerances. Further, all mandibular growth is shown to be logarithmic. As such, mandibular growth shares three logarithmic attributes: Its growth is allometric (expressed by the relative growth equation); growth of all human mandibles is capable of graphic presentation as a unitary logarithmic spiral; and its growth is gnomonic, that is, it changes its size while retaining the same shape. A synthesis of mandibular growth is presented in terms of the interrelationship between active and passive growth processes and their logarithmic attributes.

An understanding of how sutural growth processes relate to the totality of cranial growth is necessary to cure the cause and not just the symptoms of cranial synostosis. There is no direct genetic determination for the origin, growth, size, shape or maintenance of bones. Rather, phenotypic expression of skeletal tissues is regulated via genetic information encoded in the cells of functional matrices. The primary morphogenetic event in neurocranial growth is the volumetric expansion of the neural mass, which causes the surrounding neurocranial capsule to expand. Calvarial bones arise at widely separated ossification centers and spread centrifugally towards each other. Premature synostosis of the human metopic suture was noted as a frequent characteristic of the cleft-palate skull. Primary morphological event associated with premature synostosis was a cranial base malformation. Cranial sutures permit passage of the neonatal head through the birth canal, permit slight relative variations between adjacent bones while keeping these same bones relatively approximated. All morphological attributes of cranial bones and of their sutures are extrinsically determined and regulated, including both the normal and premature synostosis of the cranial sutures. It seems reasonable to the author that premature synostosis originates in the early embryonic prosencephalic head organizer, which makes it developmentally understandable.

Vascular inclusions regularly occur in the lingual dentin of the constantly erupting teeth of the pocket mouse (P. longimembris). The inclusion of a capillary loop and surrounding perivascular tissues is associated with odontoblasts whose cytodifferentiation is relatively immature. These same cells produce dentinal tubules which are more irregular in their course, more arborescent, with more lateral branches, wider in diameter and less numerous than are the tubules of the labial orthodentin. The patent vascular inclusions are surrounded by a broad halo of incompletely mineralized dentin. With further maturation complete obliteration of the vessels occurs, accompanied by complete dental matrix mineralization. A literature review supports the contention that vasodentinogenesis is related operationally to lower stages of odontoblastic cytodifferentiation although the processes by which this occurs are not yet clear.

Neurotrophism describes a non-membrane conductive activity by which interactions occur between neurons and other cells which initiate or control molecular modifications in other cells. Neuro-epithelial, neuro-muscular, and neuro-visceral trophism is defined and discussed in some detail. Some emphasis is given to data indicating possible neurotrophic control of genetic activity of muscle. It is considered that oro-facial growth may be conceived of as a homeostatically controlled series of processes in which the neural center regulates the peripheral tissues and the periphery, in turn, regulates the center. While this neurobiological hypothesis may stimulate future research, we must clearly understand that we must also inquire as to the means by which neurotrophism is integrated with those other processes involved in growth regulation.

The permanent post-canine teeth of American Whites and American Negroes of the New York City area were studied odontometrically. The mesio-distal and bucco-lingual dimensions of the crowns were measured, as was the total tooth height and crown height. The individual root lengths of these teeth were also measured, in several ways, as was root width. The mean total root volumes of these teeth was measured by a unique mercury displacement method. In another aspect of this study the mean angular divergence of the roots from a mid-coronal vertical plane was determined, while the robustness of these roots was described as a ratio between root width and length. Finally, an extensive comparison was made between our data and those in the literature on a number of African Negro populations. American Negro tooth crown and root dimensions and volumes were significantly different from those of American Whites only sporadically, although the Negro teeth usually tended to be larger. Our comparative data gave some indication that the size of both the maxillary and mandibular American negro teeth, as well as the shape of the mandibular (but not the maxillary) American Negro teeth are roughly intermediate between those of American Whites and South African Negroes. This suggestion that a “hybrid” population may possess intermediate values of crown index and of crown module is supported by our analysis of Hottentot-South African Negro hybrids with their presumptive parental stocks.

A brief review of the fundamental postulates of the method of functional cranial analysis is given, with particular emphasis on the definition of the functional matrix. Two basic types of such matrices—periosteal and capsular—are described. Periosteal matrices include muscles and teeth, while the capsular matrices are conceived of as volumes enclosed and protected by both the neurocranial and the orofacial capsules. In the neural skull the capsular matrix is the neural mass. In the facial skull this matrix is the functioning space of the oronasopharyngeal cavity. We note the following differences between the activity of periosteal and capsular functional matrices. Periosteal matrices act upon skeletal units in a direct fashion by the processes of osseous deposition and resorption (or of cartilaginous or fibrous tissue multiplication. Their net effect is to alter the form (size and shape) of their respective skeletal units. Capsular matrices act upon functional cranial components as a whole in a secondary and indirect manner. They do so by altering the volume of the capsules within which the functional cranial components are embedded. The effect of such growth changes is to cause a passive translation of these cranial components in space. Cranial growth is a combination of the morphogenetically primary activity of both types of matrix. Growth is accomplished by both spatial translation and charges in form.

Distal epiphyseal fusion of the adolescent hand was studied by frequent serial roentgenograms. A unit scoring method for quantitating epiphyseal maturation allowed direct comparison between children without regard to age, sex, or pateern of maturation. The average time for fusion of fifteen digital epiphyses of the hand was approximately 16 months, with a range of from 4 to 28 months. The rate of digital epiphyseal fusion is not correlated with sex, digital length, chronological age, or the number of epiphyses undergoing maturation. Apparently, the total time for fusion of all epiphyses is related to the time required to complete the fusion process in any given epiphysis.

The dense connective tissues of the mouse and cat are selectively stained following the vital introduction of chlorazol fast pink. In addition, the newly formed areas of bone and of cartilage are intensely stained, while other areas of these two tissues are unstained. The staining reactions of this dye parallel that of trypan blue, vitally as well as in vitro.